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The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

Uzunova SD, Zarkov AS, Ivanova AM, Stoynov SS, Nedelcheva-Veleva MN - Cell Div (2014)

Bottom Line: Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1.Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle.In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 21 "Acad. George Bonchev" Str., 1113 Sofia, Bulgaria.

ABSTRACT

Background: The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established.

Findings: This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation.

Conclusions: In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

No MeSH data available.


Related in: MedlinePlus

Independent nuclear localization of the subunits of the Tof1/Csm3/Mrc1 complex. (A) Viability test of the S-phase checkpoint GFP-tagged proteins by 10-fold serial dilution assay. 5 μL of each dilution are spotted onto YPD, YPD supplemented with 100 mM and 200 mM HU. (B-D) All GFP strains are paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins in the cell. 2.5 μg/ml DAPI staining is used for all of the probes to visualize the position of nucleus. The obtained GFP and DAPI signals are analyzed for co-localizations. GFP - Filter set 38HE (Zeiss); DAPI - Filter set 01 (Zeiss); BF – bright field.
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Figure 1: Independent nuclear localization of the subunits of the Tof1/Csm3/Mrc1 complex. (A) Viability test of the S-phase checkpoint GFP-tagged proteins by 10-fold serial dilution assay. 5 μL of each dilution are spotted onto YPD, YPD supplemented with 100 mM and 200 mM HU. (B-D) All GFP strains are paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins in the cell. 2.5 μg/ml DAPI staining is used for all of the probes to visualize the position of nucleus. The obtained GFP and DAPI signals are analyzed for co-localizations. GFP - Filter set 38HE (Zeiss); DAPI - Filter set 01 (Zeiss); BF – bright field.

Mentions: To make sure that the three S-phase checkpoint GFP-tagged proteins are fully functional, we compared their viability with that of the untagged versions. When grown on a rich YPD media, all three GFP-tagged strands revealed viability comparable to the wild-type control (Figure 1A). The GFP-tagged strands also revealed ability to withstand chronic exposure to two different concentrations of the S-phase checkpoint inducing agent HU, similar to that of the wild-type cells (Figure 1A). Then, assuming that the three GFP-tagged strains function as their untagged versions, we used them to delete a gene coding a partner subunit of the S-phase checkpoint complex Tof1/Csm3/Mrc1 that is not GFP-tagged (See Table 1, Methods). As a result, a full set of deletion mutants of the complex’s subunits was achieved. We will refer to those strains as: TOF1-GFP; csm3∆, TOF1-GFP; mrc1∆, CSM3-GFP; tof1∆, CSM3-GFP; mrc1∆, MRC1-GFP; tof1∆ and MRC1-GFP; csm3∆. Asynchronous, exponentially growing cells from the constructed strains, as well as the initial GFP strains without deletions, were paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins in the cell. DAPI DNA staining was used for all of the probes to visualize the position of nucleus. Data were documented and analyzed for all of the examined strains (Figure 1B, C, D). As was expected, the control TOF1-GFP, CSM3-GFP and MRC1-GFP strains revealed co-localization of DAPI and GFP signals, indicative of nuclear localization of the respective subunit. Interestingly, all other strains - TOF1-GFP; csm3∆, TOF1-GFP; mrc1∆, CSM3-GFP; tof1 ∆, CSM3-GFP; mrc1∆, MRC1-GFP; tof1∆ and MRC1-GFP; csm3∆, also revealed co-localization of their GFP and DAPI signals (Figure 1), suggestive of nuclear localization of the three subunits, regardless of the lack of their partners. Additionally, neither of the studied strains with deletions revealed cytoplasmic accumulation of a GFP-tagged protein. These results demonstrate that in S. cerevisiae the three subunits of the Tof1/Csm3/Mrc1 S-phase checkpoint complex are independent with regard to their nuclear translocation.


The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

Uzunova SD, Zarkov AS, Ivanova AM, Stoynov SS, Nedelcheva-Veleva MN - Cell Div (2014)

Independent nuclear localization of the subunits of the Tof1/Csm3/Mrc1 complex. (A) Viability test of the S-phase checkpoint GFP-tagged proteins by 10-fold serial dilution assay. 5 μL of each dilution are spotted onto YPD, YPD supplemented with 100 mM and 200 mM HU. (B-D) All GFP strains are paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins in the cell. 2.5 μg/ml DAPI staining is used for all of the probes to visualize the position of nucleus. The obtained GFP and DAPI signals are analyzed for co-localizations. GFP - Filter set 38HE (Zeiss); DAPI - Filter set 01 (Zeiss); BF – bright field.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4221646&req=5

Figure 1: Independent nuclear localization of the subunits of the Tof1/Csm3/Mrc1 complex. (A) Viability test of the S-phase checkpoint GFP-tagged proteins by 10-fold serial dilution assay. 5 μL of each dilution are spotted onto YPD, YPD supplemented with 100 mM and 200 mM HU. (B-D) All GFP strains are paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins in the cell. 2.5 μg/ml DAPI staining is used for all of the probes to visualize the position of nucleus. The obtained GFP and DAPI signals are analyzed for co-localizations. GFP - Filter set 38HE (Zeiss); DAPI - Filter set 01 (Zeiss); BF – bright field.
Mentions: To make sure that the three S-phase checkpoint GFP-tagged proteins are fully functional, we compared their viability with that of the untagged versions. When grown on a rich YPD media, all three GFP-tagged strands revealed viability comparable to the wild-type control (Figure 1A). The GFP-tagged strands also revealed ability to withstand chronic exposure to two different concentrations of the S-phase checkpoint inducing agent HU, similar to that of the wild-type cells (Figure 1A). Then, assuming that the three GFP-tagged strains function as their untagged versions, we used them to delete a gene coding a partner subunit of the S-phase checkpoint complex Tof1/Csm3/Mrc1 that is not GFP-tagged (See Table 1, Methods). As a result, a full set of deletion mutants of the complex’s subunits was achieved. We will refer to those strains as: TOF1-GFP; csm3∆, TOF1-GFP; mrc1∆, CSM3-GFP; tof1∆, CSM3-GFP; mrc1∆, MRC1-GFP; tof1∆ and MRC1-GFP; csm3∆. Asynchronous, exponentially growing cells from the constructed strains, as well as the initial GFP strains without deletions, were paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins in the cell. DAPI DNA staining was used for all of the probes to visualize the position of nucleus. Data were documented and analyzed for all of the examined strains (Figure 1B, C, D). As was expected, the control TOF1-GFP, CSM3-GFP and MRC1-GFP strains revealed co-localization of DAPI and GFP signals, indicative of nuclear localization of the respective subunit. Interestingly, all other strains - TOF1-GFP; csm3∆, TOF1-GFP; mrc1∆, CSM3-GFP; tof1 ∆, CSM3-GFP; mrc1∆, MRC1-GFP; tof1∆ and MRC1-GFP; csm3∆, also revealed co-localization of their GFP and DAPI signals (Figure 1), suggestive of nuclear localization of the three subunits, regardless of the lack of their partners. Additionally, neither of the studied strains with deletions revealed cytoplasmic accumulation of a GFP-tagged protein. These results demonstrate that in S. cerevisiae the three subunits of the Tof1/Csm3/Mrc1 S-phase checkpoint complex are independent with regard to their nuclear translocation.

Bottom Line: Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1.Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle.In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 21 "Acad. George Bonchev" Str., 1113 Sofia, Bulgaria.

ABSTRACT

Background: The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established.

Findings: This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation.

Conclusions: In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

No MeSH data available.


Related in: MedlinePlus